The present invention relates to a process for the production of a mammalian stromelysin catalytic domain protein, to a purified mammalian stromelysin catalytic domain protein, to pharmaceutical compositions which include the mammalian stromelysin catalytic domain protein and a pharmaceutically acceptable carrier, and to pharmaceutical methods of treatment.
Matrix metalloproteinases, such as stromelysins, collagenases, and gelatinases, are believed to be involved in connective tissue degradation (Woessner J F, FASEB J. 1991;5:2145-2154) in several physiological and pathological processes including the cartilage degradation in arthritis and tumor progression and metastasis (McDonnell S, Matrisian L, Cancer Metastasis Rev. 1990;9:305-319). Therefore, there is great interest in understanding the catalytic mechanism of these matrix metalloproteinases and designing specific inhibitors to control their activity.
Similar to other matrix metalloproteinases, the human fibroblast stromelysin (Whitham S E, Murphy G, Angel P, et al, Biochem. J. 1986;240:913-916; Saus J, Quinones S, Otani Y, et al, J. Biol. Chem. 1988;263:6742-6745) has a signal peptide for secretion, a propeptide with a cysteine residue for maintaining latency (Van Wart H E, Birkedal-Hansen H, Proc. Natl. Acad. Sci. U.S.A. 1990;87:5578-5582; Park A J, Matrisian L M, Kells A F, et al, J. Biol. Chem. 1991;266:1584-1590), a catalytic domain with a conserved sequence highly homologous with the zinc binding site in the bacterial zinc proteinase thermolysin (Vallee B L, Auld D S, Biochemistry 1990;29:5647-5659), and a C-terminal fragment which may be involved in substrate and inhibitor binding (Allan J A, Hembry R M, Angal S, et al, J. Cell Sci. 1991;99:789-795; Murphy G, Allan J A, Willenbrock F, et al, J. Biol. Chem. 1992;267:9612-9618). The matrix metalloproteinases are all secreted as proenzymes and are activated in vivo by a mechanism not yet determined. However, these enzymes can be activated in vitro with organomercurials, proteolytic enzymes, chaotropic agents, or heat (Okada Y, Harris E D, Nagase H, Biochem. J. 1988;254:731-741; Nagase H, Enghild J J, Suzuki K, Salvesen G, Biochemistry 1990;29:5783-5789; Koklitis P A, Murphy G, Sutton C, Angal S, Biochem. J. 1991;276:217-221). Removal of the propeptide from prostromelysin by proteinases and organomercurial compounds is a stepwise process (Okada, supra, 1988; Nagase, supra, 1990) which generates intermediate forms before the propeptide is removed completely by activated stromelysin. The activated enzyme undergoes autolytic cleavage at sites close to the C-terminus, producing a 28-kDa fragment as well as smaller species (Okada, supra, 1988; Koklitis, supra, 1991). The instability of matrix metalloproteinases due to the autodegradation may partially account for the difficulty in structural determination by X-ray crystallography. Matrilysin (formerly called PUMP) is a unique member of this enzyme family in that it lacks the C-terminal portion found in stromelysins, collagenases, and gelatinases (Muller D, Quantin B, Gesnel M-C, et al, Biochem. J. 1988;253:187-192). C-Terminal-deleted stromelysin and collagenase have been made and they have shown activity similar to the full-length enzymes (Marcy A I, Eiberger L L, Harrison R, et al, Biochemistry 1991;30:6476-6483; Lowry C L, McGeehan G, LeVine H I, Proteins: Struct., Funct., Genet. 1992;12:42-48; Murphy, supra, 1992). Thus, Marcy, supra, 1991, expressed a truncated stromelysin containing the catalytic domain and the propeptide. The propeptide was removed in vitro to generate the catalytic domain (Marcy, supra, 1991; Salowe S P, Marcy A I, Cuca G C, et al, Biochemistry 1992;31:4535-4540). Lowry, supra, 1992, described a stability study using a recombinant 19-kDa collagenase catalytic domain. However, the expression and purification of the collagenase catalytic domain have not been described. More recently, Murphy, supra, 1992, expressed the C-terminal-deleted procollagenase and prostromelysin in mouse cells. Therefore, the N-terminal catalytic domain of stromelysin is responsible and sufficient for the proteinase activity, and the C-terminal portion can be removed without major modification to the active site of the catalytic domain.
As mentioned above, matrix metalloproteinases share high sequence homology. Catalytic domains in each of the metalloproteinases can be identified by sequence comparison as described in the literature (Murphy G J P, et al, FEBS 1991;289:4-7; Muller, supra, 1988; Woessner, supra, 1991).
The object of the present invention is the expression, purification, and characterization of a 20-kDa stromelysin catalytic domain (SCD) protein lacking both the propeptide and the C-terminal fragment (FIG. 1). The removal of the propeptide eliminates the need for proteolytic or chemical activation, and the removal of the C-terminal fragment removes autolytic sites, thereby making the protein resistant to autodegradation. The active and stable protein with a mass of 20-kDa is suitable for structure determination by nuclear magnetic resonance spectroscopy and X-ray crystallography, as well as mechanistic studies of catalysis and inhibition. Additionally, the protein is useful in the therapy of various disease states.